Acquired linezolid resistance in DR-TB: genotype-phenotype discordance and molecular heterogeneity in a retrospective cohort

Linezolid is an important drug for the treatment of drug-resistant tuberculosis (DR-TB). Acquired linezolid resistance threatens treatment efficacy. This study aimed to analyze the risk factors and molecular mechanisms of linezolid-acquired resistance in a clinical cohort. We conducted a retrospective study of 61 patients with DR-TB who failed linezolid-containing regimens (2017–2021). Paired baseline and post-treatment Mycobacterium tuberculosis isolates were subjected to linezolid Minimum inhibitory concentration (MIC) testing (Microplate Alamar Blue Assay) and sequencing of rplC, rrl (23 S rRNA), and rplD. Acquired resistance was defined as a ≥ 4-fold increase in MIC or the presence of new resistance-conferring mutation. Risk factors were analyzed using logistic regression. Acquired linezolid resistance occurred in 31.1% (19/61) of patients. Younger age (< 37.5 years; OR = 1.04/year decrease, p = 0.012) and prolonged therapy (> 14.3 months; OR = 1.07/month, p = 0.004) were the independent risk factors. Sequencing revealed 26 mutation events across the longitudinal isolates; 57.7% (15/26) harbored rplC T460C (Cys154Arg), whereas 38.5% (10/26) had rrl mutations (G2270T, G2814T). A novel rplD A518T (Lys173Ile) mutation emerged concurrently with the rplC T460C mutation. Phenotypic-genotypic discordance occurred in 11.5% (3/26) of resistant isolates, including one strain with MIC elevation (0.25→4.0 mg/L) lacking target mutations. Linezolid resistance correlated with prior resistance to clofazimine (kappa = 0.538, p < 0.001), amikacin, and pasiniazid. Young age, extended therapy, and ribosomal mutations drive linezolid resistance. Pretreatment susceptibility screening is critical.